Abnormalities in pancreatic beta-cell insulin secretion mechanisms can lead to diabetes. However, metabolic regulation of insulin exocytosis is only partly understood. Proximal signaling events have been relatively well defined, but less is known about molecular events that control insulin exocytosis. Increased beta-cell nutrient metabolism leads to a rapid rise in cytosolic [Ca2+]i, considered to be a major secondary signal for stimulating insulin release. However, it is unclear how [Ca2+]i triggers the beta-cell's exocytotic machinery at the molecular level. A long-term objective is to better define the molecular mechanism underlying Ca2+-dependent control of insulin exocytosis. In order to gain a 'handle' on the beta-cell's exocytotic machinery, we have focused on the beta-granule protein, Rab3A. In beta-cells, Rab3A interacts with calmodulin in a GTP- and Ca2+-dependent manner. This provides a means for calmodulin to be recruited to a beta-granule, but as local cytosolic [Ca2+]i increases it is transferred to other calmodulin binding proteins (e.g. CaMK-II, PP-2B etc.) for their specific activation on a beta-granule and/or site of exocytosis. This led us to investigate Ca2+-dependent phosphorylation of beta-granule membrane proteins. Surprisingly, the major beta-granule phosphoproteins are dephosphorylated in response to [Ca2+]i, implicating a novel role for PP-2B (a.k.a. calcineurin) in control insulin release. One of these beta-granule proteins was kinesin which when phosphorylated has its motor activity and ability to attach vesicles to microtubules inhibited. Thus, Ca2+-induced PP-2B mediated dephosphorylation of kinesin increased beta-granule transport. This represents a first insight into a link between a secondary coupling signal and a component of the exocytotic mechanism in the beta-cell. However, a Rab3A-calmodulin interaction likely controls other steps in the insulin exocytosis mechanism. Intriguingly, we have recently found that Rab3A knockout mice develop diabetes, due to insulin secretory dysfunction of the beta-cell, but this phenotype needs further characterization which should also better define the role of Rab3A in insulin exocytosis. Recombinant adenoviruses to express PP-2B and a specific PP-2B inhibitor, CAIN, have been obtained to better examine the role of PP-2B in control of insulin exocytosis. Moreover, there are two other PP-2B phosphoprotein substrates on the beta-granule to be identified and characterized (p42 and p36). Notwithstanding, [Ca2+]i controls other steps in the insulin exocytosis mechanism independently of Rab3A/PP2B, particularly at the distal stages in forming an exocytotic membrane-fusion pore. In this regard, two candidate Ca2+-binding secretory granule proteins, synaptotagmin- 3 and syncollin, are under investigation in control of insulin exocytosis. Adenoviruses that express various forms of synaptotagmin-3 and syncollin have been generated, and a syncollin-like protein in beta-granules (p10) identified. An adenovirus expressing a syncollin-GFP fusion protein enables us to track beta-granule motion/exocytosis. It is intended that new insight into Ca2+-dependent control of insulin exocytosis will be gained from these proposed studies that, in turn, can lead to novel diabetes therapies.